Centromeres play a critical role in chromosome and chromatid segregation in meiosis and mitosis. Abnormal centromeric structure or function may play a role in both nondisjunction and simple chromosome loss. Virtually nothing is known about the sequence organization of centromeric DNA in any organism except yeast, in which there is a distinctive 112-119 bp centromeric sequence. There are only minor differences in this sequence in different yeast chromosomes. We have found a repetitive sequence element within a cloned 2.3 kb human DNA fragment that shares several features of sequence organization with yeast centromeric DNA and also hybridizes to thee centromeric region of every human chromosome, even under stringent conditions. We intend to subclone the single 170 bp repeat (of 14 in the cloned fragment) that contains this sequence, to isolate homologous sequences from several different human chromosomes and to find out if there is a consensus sequence for this putative centromeric DNA. These sequences will be tested for centromeric function in yeast and mammalian cells. The nuclease sensitivity of the 170 bp subcloned fragment in chromatin will be determined, because yeast contromeres in chromatin are nuclease resistant. The cloned 2.3 kb human DNA fragment is known to be closely associated with the heterochromatic repeated DNA sequences of some human chromosomes. We intend to isolate a junction fragment, to examine its DNA sequence. Centromeric DNA in the mouse will be isolated and characterized to look for features shared by centromeric sequences in human and mouse. A mouse minor satellite was recently described which contains a 9 bp element partially homologous to the highly conserved 8 bp CDEI region of yeast centromeric DNA. Stable plasmid clones containing tandem repeats of this 130 bp minor satellite will be isolated from mouse DNA, sequenced and a sequence organization corresponding to that of human and yeast centromeric DNA looked for.